PEGylation by the Dock and Lock (DNL) Technique

ABSTRACT

The present invention concerns methods and compositions for forming PEGylated complexes of defined stoichiometry and structure. In preferred embodiments, the PEGylated complex is formed using dock-and-lock technology, by attaching a target agent to a DDD sequence and attaching a PEG moiety to an AD sequence and allowing the DDD sequence to bind to the AD sequence in a 2:1 stoichiometry, to form PEGylated complexes with two target agents and one PEG moiety. In alternative embodiments, the target agent may be attached to the AD sequence and the PEG to the DDD sequence to form PEGylated complexes with two PEG moieties and one target agent. In more preferred embodiments, the target agent may comprise any peptide or protein of physiologic or therapeutic activity. The PEGylated complexes exhibit a significantly slower rate of clearance when injected into a subject and are of use for treatment of a wide variety of diseases.

RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 11/925,408, filed Oct. 26, 2007, which is a continuation-in-part ofU.S. patent application Ser. No. 11/391,584 (now issued U.S. Pat. No.7,521,056), filed Mar. 28, 2006, which claimed the benefit of U.S.Provisional Patent Applications 60/668,603 (now expired), filed Apr. 6,2005; 60/728,292 (now expired), filed Oct. 20, 2005; 60/751,196 (nowexpired), filed Dec. 16, 2005; and 60/782,332 (now expired), filed Mar.14, 2006; and is a continuation-in-part of U.S. patent application Ser.No. 11/478,021 (now issued U.S. Pat. No. 7,534,866), filed Jun. 29,2006, which claimed the benefit of U.S. Provisional Patent Applications60/728,292 (now expired), filed Oct. 20, 2005; 60/751,196 (now expired),filed Dec. 16, 2005; and 60/782,332 (now expired), filed Mar. 14, 2006;and is a continuation-in-part of U.S. patent application Ser. No.11/633,729 (now issued U.S. Pat. No. 7,527,787), filed Dec. 5, 2006,which was a continuation-in-part of PCT/US06/10762, filed Mar. 24, 2006;PCT/US06/12084, filed Mar. 29, 2006; PCT/US06/25499, filed Jun. 29,2006; U.S. Ser. No. 11/389,358 (now issued U.S. Pat. No. 7,550,143),filed Mar. 24, 2006; Ser. No. 11/391,584 (now issued U.S. Pat. No.7,521,056), filed Mar. 28, 2006; and Ser. No. 11/478,021 (now issuedU.S. Pat. No. 7,534,866), filed Jun. 29, 2006; and claimed the benefitof U.S. Provisional Patent Applications 60/751,196 (now expired), filedDec. 16, 2005; and 60/864,530 (now expired), filed Nov. 6, 2006; thetext of each cited application incorporated herein by reference in itsentirety.

BACKGROUND

The efficacy of a therapeutic agent may be enhanced by improving itsbioavailability via several means, one of which is PEGylation, a processof chemically linking polyethylene glycol (PEG) to the therapeutic agentof interest, with the resulting conjugate exhibiting an increased serumhalf-life. Additional advantages of the PEGylated products may alsoinclude lower immunogenicity, decreased dosing frequency, increasedsolubility, enhanced stability, and reduced renal clearance. Because themost common reactive sites on proteins (including peptides) forattaching PEG are the ε amino groups of lysine and the α amino group ofthe N-terminal residue, early methods of PEGylation resulted inmodification of multiple sites, yielding not only monoPEGylatedconjugates consisting of mixtures of positional isomers, such asPEGINTRONT™ (Grace et al., J. Biol. Chem. 2005; 280:6327) and PEGASYS®(Dhalluin et al., Bioconjugate Chem. 2005; 16:504), but also adductscomprising more than one PEG chain. Site-specific attachment of a singlePEG to the α amino group of the N-terminal residue was reported to bethe predominant product upon reacting PEG-aldehyde (PEG-ALD) at low pHwith IFN-β1b (Basu et al., Bioconjugate Chem. 2006; 17:618) or IFN-β1a(Pepinsky et al., J. Pharmacol. Exp. Ther. 2001; 297:1059). Similarstrategies were applied to prepare N-terminally linked PEG to G-CSF(Kinstler et al., Pharm. Res. 1996; 13:996) or type I soluble tumornecrosis factor receptor (Kerwin et al., Protein Sci. 2002; 11:1825).More recently, a solid-phase process for PEGylation of the N-terminus ofrecombinant interferon alpha-2a was reported (Lee et al., Bioconjug.Chem. Oct. 18, 2007, epub).

Site-directed PEGylation of a free cysteine residue introduced into atarget protein has also been achieved with PEG-maleimide (PEG-MAL) forseveral recombinant constructs including IL-2 (Goodson and Katre,Biotechnology. 1990:8:343); IFN-α2 (Rosendahl et al., Bioconjugate Chem.2005; 16:200); GM-CSF (Doherty et al., Bioconjugate Chem. 2005;16:1291); scFv (Yang et al., Protein Eng. 2003; 16:761), andminiantibodies (Kubetzko et al., J. Biol. Chem; 2006; 201:35186). Apopular approach for improving the therapeutic efficacy of an enzyme hasbeen to prepare conjugates containing multiple PEG of small size, asknown for methioninase (Yang et al., Cancer Res. 2004; 64:6673);L-methione γ-lyase (Takakura et al., Cancer Res. 2006:66:2807): argininedeaminase (Wang et al., Bioconjugate Chem. 2006; 17:1447); adenosinedeaminase (Davis et al., Clin. Exp. Immunol. 1981; 46:649);L-asparaginase (Bendich et al., Clin. Exp. Immunol. 1982; 48:273); andliver catalase (Abuchowski et al., J. Biol. Chem. 1977; 252:3582).

PEGylations of bovine serum albumin (Abuchowski et al., J. Biol. Chem.1977; 252:3578); hemoglobin (Manjula et al., Bioconjugate Chem. 2003;14:464); visomant (Mosharraf et al., Int. J. Pharm. 2007; 336:215);small molecules such as inhibitors of integrin α4β1 (Pepinsky et al., J.Pharmacol. Exp. Ther. 2005; 312:742); lymphoma-targeting peptides(DeNardo et al., Clin. Cancer. Res. 2003; 9(Suppl.):3854s); anti-VEGFaptamer (Bunka and Stockley, Nat. Rev. Microbiol. 2006; 4:588) andoligodeoxynucleotides (Fisher et al., Drug Metab. Dispos. 2004; 32:983)have also been described. However, there exists a need for a generalmethod of PEGylation that would produce exclusively a monoPEGylatedconjugate composed of a single PEG linked site-specifically to apredetermined location of the candidate agent and retains thebioactivity of the unmodified counterpart.

SUMMARY OF THE INVENTION

The present invention discloses methods and compositions for producingPEGylated compounds with selected numbers of attached PEG residues thatare attached at selected locations of a candidate agent. In preferredembodiments, the agents are monoPEGylated. In more preferredembodiments, the target to be PEGylated may be attached to a DDD(dimerization and docking domain) sequence and a PEG moiety may beattached to an AD (anchor domain) sequence as described in more detailbelow. Dimers of the DDD sequence bind with high affinity to monomers ofthe AD sequence, resulting in formation of a monoPEGylated target agentdimer. The stoichiometry of binding and location of the PEG residue aredetermined by the specificity of the DDD/AD interaction.

In more preferred embodiments, the monoPEGylated complex may becovalently stabilized by introduction of cysteine residues atappropriate locations in the DDD and AD sequences, to form disulfidebonds that stabilize the complex. In other embodiments, the PEG reagentsmay be capped at one end with a linear or branched methoxy group(m-PEG).

In other preferred embodiments, the PEGylated complex made by the DNLmethod shows a rate of clearance from serum that is at least an order ofmagnitude slower than the unPEGylated target agent. In certainalternative embodiments, the PEGylated complex may be alternativelyconstructed with the PEG moiety attached to the DDD sequence and thetarget agent attached to the AD sequence, resulting in a stoichiometryof 2 PEG to 1 target agent per complex.

The skilled artisan will realize that virtually any physiologically ortherapeutically active agent to be administered in vivo may bestabilized by PEGylation, including but not limited to enzymes,cytokines, chemokines, growth factors, peptides, apatamers, hemoglobins,antibodies and fragments thereof. Exemplary agents include MIF, HMGB-1(high mobility group box protein 1), TNF-α, IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16,IL-17, IL-18, IL-19, IL-23, IL-24, CCL19, CCL21, IL-8, MCP-1, RANTES,MIP-1A, MIP-1B, ENA-78, MCP-1, IP-10, Gro-β, Eotaxin, interferon-α, -β,-80 , G-CSF, GM-CSF, SCF, PDGF, MSF, Flt-3 ligand, erythropoietin,thrombopoietin, hGH, CNTF, leptin, oncostatin M, VEGF, EGF, FGF, PlGF,insulin, hGH, calcitonin, Factor VIII, IGF, somatostatin, tissueplasminogen activator, and LIF.

The monoPEGylated complexes, are suitable for use in a wide variety oftherapeutic and diagnostic applications. Methods of use of monoPEGylatedcomplexes may include detection, diagnosis and/or treatment of a diseaseor other medical condition. Such conditions may include, but are notlimited to, cancer, hyperplasia, diabetes, diabetic retinopathy, maculardegeneration, inflammatory bowel disease, Crohn's disease, ulcerativecolitis, rheumatoid arthritis, sarcoidosis, asthma, edema, pulmonaryhypertension, psoriasis, corneal graft rejection, neovascular glaucoma,Osler-Webber Syndrome, myocardial angiogenesis, plaqueneovascularization, restenosis, neointima formation after vasculartrauma, telangiectasia, hemophiliac joints, angiofibroma, fibrosisassociated with chronic inflammation, lung fibrosis, deep venousthrombosis or wound granulation.

In particular embodiments, the disclosed methods and compositions may beof use to treat autoimmune disease, such as acute idiopathicthrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura,dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupuserythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, juvenile diabetes mellitus,Henoch-Schonlein purpura, post-streptococcal nephritis, erythemanodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis,multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis, psoriasis or fibrosingalveolitis.

It is anticipated that any type of tumor and any type of tumor antigenmay be targeted. Exemplary types of tumors that may be targeted includeacute lymphoblastic leukemia, acute myelogenous leukemia, biliarycancer, breast cancer, cervical cancer, chronic lymphocytic leukemia,chronic myelogenous leukemia, colorectal cancer, endometrial cancer,esophageal, gastric, head and neck cancer, Hodgkin's lymphoma, lungcancer, medullary thyroid cancer, non-Hodgkin's lymphoma, multiplemyeloma, renal cancer, ovarian cancer, pancreatic cancer, glioma,melanoma, liver cancer, prostate cancer, and urinary bladder cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Cartoon illustration of the DNL method. Triangles depictcomponent A, which forms a homodimer (a₂) mediated by the dimerizationand docking domain (DDD). The location of free thiol groups (SH) of theengineered cysteine residues is indicated. Octagons depict component Bcontaining an anchor domain (AD) peptide. The DNL reaction results inthe generation of a covalent trimeric structure via binding of DDD andAD peptides and subsequent formation of disulfide bridges.

FIG. 2. Cartoon drawing of IMP362. A 20 kDa PEG (starburst), AD2 peptide(helix), EDANS fluorescent tag (oval) and the positions of freesulfhydryl groups (SH) are indicated.

FIG. 3. Cartoon drawing of IMP413. A 30 kDa PEG (starburst), AD2 peptide(helix), EDANS fluorescent tag (oval) and the positions of freesulfhydryl groups (SH) are indicated.

FIG. 4. Analysis of roller bottle production and purification byanti-IFNa immunoblot and ELISA. Samples were diluted as indicated and 5μl were subjected to reducing SDS-PAGE and immunoblot analysis withpolyclonal anti-IFNα. The dilution, total volume and fraction analyzedof the total volume (f) for each sample is given. The amount of proteinin each band was estimated from standards and divided by f to give thetotal protein estimate. Total protein measurements determined by ELISAare also given.

FIG. 5. Reducing SDS-PAGE analysis of IFNα2b-DDD2 following IMACpurification with Ni-NTA. Two independent preparations of IFNα2b-DDD2(lanes 1 &2), which were eluted from Ni-NTA resin with 250 mM imidazolebuffer, and the 30 mM imidazole buffer column wash were resolved bySDS-PAGE under reducing conditions and stained with Coomassie blue. Theposition of M_(r) standards and IFNα2b-DDD2 (arrow) are indicated.

FIG. 6. Cartoon drawing of α2b-362. IFNα2b groups (pentagons), 20 kDaPEG (starburst), AD2 and DDD2 peptides (helices) and EDANS fluorescenttag (oval) are indicted.

FIG. 7. Analysis of CM-FF IEC purification of IFNα2b-DDD2-IMP362 bySDS-PAGE with Coomassie Blue staining (A), direct fluorescence imaging(B) and anti-IFNα immunoblotting (C). The same gel was used forfluorescence imaging and subsequent Coomassie blue staining. For thisgel (A+B), all fractions were concentrated to correspond to the reactionvolume (3.5 mL) and 5 μl/lane were loaded. Both reduced and non-reducedsamples were run as indicated. For the immunoblot (C), all samples werediluted 1:50 from those used for gel A/B. Solid black, grey and openarrow-heads show the positions of IFNα2b-DDD2-IMP362, IFNα2b-DDD2 andIMP362, respectively. The positions of M_(r) standards are alsoindicated.

FIG. 8. Cartoon drawing of α2b-413. IFNα2b groups (pentagons), 30 kDaPEG (starburst), AD2 and DDD2 peptides (helices) and EDANS fluorescenttag (oval) are indicted.

FIG. 9. Dose-response curves showing in vitro growth inhibition ofBurkitt's lymphoma (Daudi) cells after 4-days in culture in the presenceof either rhIFN-α2b standard, IFN-α2b-DDD2 or α2b-362. MTS dye was addedto the plates, which were incubated for 3 h before measuring the OD₄₉₀.The % of the signal obtained from untreated cells was plotted vs. thelog of the molar concentration. The 50% effective concentration (EC₅₀)values were obtained by sigmoidal fit non-linear regression using GraphPad Prism software.

FIG. 10. Evaluation of the pharmacokinetic properties of IFNαconstructs. Each reagent (test and control) was administered toSwiss-Webster mice at equimolar protein doses as a single bolus i.v.injection of 3 μg for rhuIFN-α2a, 5 μg for PEGINTRONT™, 11 μg forα2b-362, and 13 μg for α2b-413. Serum samples were isolated at the timesindicated and the serum concentrations of IFN-αwere determined by ELISA.The ρM concentration was plotted vs. hours post injection. Datarepresents the mean value from two mice.

FIG. 11. Evaluation of the therapeutic efficacy of IFNα constructs inmice bearing Burkitt's lymphoma (Daudi). Eight-week-old female SCID micewere injected i.v. with 1.5×10⁷ Daudi cells. Groups of 5 mice wereadministered PEGINTRON™, α2b-362 and α2b-413 at doses of 3,500, 7,000 or14,000 Units once per week for 4 weeks. Therapy commenced 1 day afterthe Daudi cells were transplanted. Injection times are indicated witharrows. Survival curves and median survival are shown for each group.

FIG. 12. Evaluation of the dosing schedule for therapy of tumor-bearingmice. Eight-week-old female SCID mice were injected i.v. with 1.5×10⁷Daudi-cells. Groups of 6-7 mice were administered 14,000 IU of eitherPEGINIRONT™ or α2b-413 via a s.c. injection. Therapy was commenced 1 dayafter the Daudi cells were administered to the mice. Groups were dosedonce a week (q7dx4), once every other week (q2wkx4) or once every 3weeks (q3wkx4). Injection times are indicated with arrows. All the micereceived 4 injections in total. Survival curves and median survival areshown for each group.

FIG. 13. Cartoon drawing of G-CSF-413. G-CSF groups (pentagons), 30 kDaPEG (starburst), AD2 and DDD2 peptides (helices) and EDANS fluorescenttag (oval) are indicted.

FIG. 14. SDS-PAGE and anti-EPO immunoblot analysis of IMAC-purifiedEPO-DDD2. Proteins were resolved by SDS-PAGE under reducing ornon-reducing conditions and gels were stained with Coomassie blue ortransferred to PVDF membranes for immunoblot analysis with an anti-EPOmonoclonal antibody. The amounts of protein loaded/lane is indicated atthe bottom of the lanes. The positions of MW standards and of EPO-DDD2(arrow) are indicated.

FIG. 15. Cartoon drawings of h679-Fab-DDD2 (A), dimeric EPO-DDD2 (B),which combine to create EPO-679 (C) by the DNL method. The variable andconstant domains of h679 Fab (ovals), AD2 and DDD2 helices, EPO groups(pentagons) and free sulthydryl groups (SH) are indicated.

FIG. 16. SDS-PAGE analysis of EPO-679. Proteins (1 μg) were resolved bySDS-PAGE under reducing and non-reducing conditions in the lanes asindicated and gels were stained with Coomassie blue. The positions of MWstandards and all relevant species (arrows) are indicated.

FIG. 17. Stimulation of cell growth by EPO-constructs. EPO-responsiveTF1 cells (1×10⁴) were cultured for 72 hours in the presence of rhEPO,EPO-DDD2 or EPO-679. The relative viable cell density was determined byMTS assay. The log of the concentration in U/mL is plotted vs. OD₄₉₀.

FIG. 18. Cartoon drawing of EPO-413. EPO groups (pentagons), 30 kDa PEG(starburst), AD2 and DDD2 peptides (helices) and EDANS fluorescent tag(oval) are indicted.

Dock and Lock (DNL) Method

The key to the DNL method is the exploitation of the specificprotein/protein interactions occurring in nature between the regulatory(R) subunits of cAMP-dependent protein kinase (PKA) and the anchoringdomain (AD) of A-kinase anchoring proteins (AKAPs) (Baillie et al., FEBSLetters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol.2004; 5: 959). PKA, which plays a central role in one of the beststudied signal transduction pathway triggered by the binding of thesecond messenger cAMP to the R subunits, was first isolated from rabbitskeletal muscle in 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763).The structure of the holoenzyme consists of two catalytic subunits heldin an inactive form by the R subunits (Taylor, J. Biol. Chem. 1989;264:8443). Isozymes of PKA are found with two types of R subunits (RIand RII), and each type has α and β isoforms (Scott, Pharmacol. Ther.1991; 50:123). The R subunits have been isolated only as stable dimersand the dimerization domain has been shown to consist of the first 44amino-terminal residues (Newlon et al., Nat. Struct. Biol. 1999; 6:222).Binding of cAMP to the R subunits leads to the release of activecatalytic subunits for a broad spectrum of serine/threonine kinaseactivities, which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). Interestingly, AKAPs will only bind to dimeric Rsubunits. For human RIIα, the AD binds to a hydrophobic surface formedby the 23 amino-terminal residues (Colledge and Scott, Trends Cell Biol.1999; 6:216). Thus, the dimerization domain and AKAP binding domain ofhuman Ma are both located within the same N-terminal 44 amino acidsequence (Newton et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al.,EMBO J. 2001; 20:1651), which is termed the DDD herein.

DDD of Human RIIα and AD of AKAPs as Linker Modules

We have developed a platform technology to utilize the DDD of human RIIαand the AD of a certain amino acid sequence as an excellent pair oflinker modules for docking any two entities, referred to hereafter as Aand B, into a noncovalent complex, which could be further locked into astably tethered structure through the introduction of cysteine residuesinto both the DDD and AD at strategic positions to facilitate theformation of disulfide bonds, as illustrated in FIG. 1. The generalmethodology of the “dock-and-lock” approach is as follows. Entity A isconstructed by linking a DDD sequence to a precursor of A, resulting ina first component hereafter referred to as a. Because the DDD sequencewould effect the spontaneous formation of a dimer, A would thus becomposed of a₂. Entity B is constructed by linking an AD sequence to aprecursor of B, resulting in a second component hereafter referred to asb. The dimeric motif of DDD contained in a₂ will create a docking sitefor binding to the AD sequence contained in b, thus facilitating a readyassociation of a₂ and b to form a binary, trimeric complex composed ofa₂b. This binding event is made irreversible with a subsequent reactionto covalently secure the two entities via disulfide bridges, whichoccurs very efficiently based on the principle of effective localconcentration because the initial binding interactions should bring thereactive thiol groups placed onto both the DDD and AD into proximity(Chimura et al., Proc. Natl. Acad. Sci. USA. 2001; 98:8480) to ligatesite-specifically.

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,nucleic acids, and PEG. The DNL method was disclosed in each of thefollowing U.S. Provisional patent applications: 60/728,292, filed Oct.20, 2005; 60/751,196, filed Dec. 16, 2005; and 60/782,332, filed Mar.14, 2006, and U.S. patent application Ser. No. 11/389,358, allincorporated herein by reference in their entirety.

PEGylation by DNL

In a preferred method, the target to be PEGylated is linked to a DDDsequence to generate the DDD module. A PEG reagent of a desirablemolecular size is derivatized with a related AD sequence and theresulting PEG-AD module is combined with the DDD module to produce thePEGylated conjugate that consists of a single PEG tetheredsite-specifically to two copies of the target via the disulfide bondsformed between DDD and AD. The PEG reagents are capped at one end with amethoxy group (m-PEG), can be linear or branched, and may contain one ofthe following functional groups: propionic aldehyde, butyric aldehyde,ortho-pyridylthioester (OPTE), N-hydroxysuccinimide (NHS),thiazolidine-2-thione, succinimidyl carbonate (SC), maleimide, orortho-pyridyldisulfide (OPPS). Among the targets that may be of interestfor PEGylation are enzymes, cytokines, chemokines, growth factors,peptides, aptamers, hemoglobins, antibodies and fragments. The method isnot limiting and a wide variety of agents may be PEGylated using thedisclosed methods and compositions.

EXAMPLES

The following examples are provided to illustrate, but not to limit, theclaims of the present invention.

Example 1 Generation of PEG-AD2 Modules

Synthesis of IMP350

CGQIEYLAKQIVDNAIQQAGC(SS-tbu)-NH₂ (SEQ ID NO: 1) MH⁺ 2354

IMP350 was made on a 0.1 mmol scale with Sieber Amide resin using Fmocmethodology on a Protein Technologies PS3 peptide synthesizer. Startingfrom the C-terminus the protected amino acids used wereFmoc-Cys(t-Buthio)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH,Fmoc-Asp(OBut)-OH, Fmoc-Val-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Tyr(But)-OH,Fmoc-Glu(OBut)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH andFmoc-Cys(Trt)-OH. The peptide was cleaved from the resin and purified byreverse phase (RP)-HPLC.

Synthesis of PEG₂₀-IMP350

IMP350 (0.0104 g) was mixed with 0.1022 g of mPEG-OPTE (20 kDa, NektarTherapeutics) in 7 mL of 1 M Tris buffer at pH 7.81. Acetonitrile, 1 mL,was then added to dissolve some suspended material. The reaction wasstirred at room temperature for 3 h and then 0.0527 g of TCEP was addedalong with 0.0549 g of cysteine. The reaction mixture was stirred for1.5 h and then purified on a PD-10 desalting column, which wasequilibrated with 20% methanol in water. The sample was eluted, frozenand lyophilized to obtain 0.0924 g of crude PEG₂₀-IMP350 (MH+ 23508 byMALDI).

Synthesis of IMP360

CGQIEYLAKQIVDNAIQQAGC(SS-tbu)G-EDANS (SEQ ID NO: 1) MH⁺ 2660

IMP 360 was synthesized on a 0.1 mmol scale with EDANS resin (NovaBiochem) using Fmoc methodology on a Protein Technologies PS3 peptidesynthesizer. The Fmoc-Gly-OH was added to the resin manually using 0.23g of Fmoc-Gly-OH, 0.29 g of HATU, 26 μL of DIEA, 7.5 mL of DMF and 0.57g of EDANS resin (Nova Biochem). The reagents were mixed and added tothe resin. The reaction was mixed at room temperature for 2.5 hr and theresin was washed with DMF and IPA to remove the excess reagents.Starting from the C-terminus the protected amino acids used wereFmoc-Cys(t-Buthio)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH,Fmoc-Asp(OBut)-OH, Fmoc-Val-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Tyr(But)-OH,Fmoc-Glu(OBut)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH andFmoc-Cys(Trt)-OH. The peptide was cleaved from the resin and purified byRP-HPLC.

Synthesis of IMP362 (PEG₂₀-IMP360)

A cartoon diagram of IMP362 is provided in FIG. 2. For synthesis ofIMP362, IMP360 (0.0115 g) was mixed with 0.1272 g of mPEG-OPTE (20 kDa,Nektar Therapeutics) in 7 mL of 1 M tris buffer, pH 7.81. Acetonitrile(1 mL) was then added to dissolve some suspended material. The reactionwas stirred at room temperature for 4 h and then 0.0410 g of TCEP wasadded along with 0.0431 g of cysteine. The reaction mixture was stirredfor 1 h and purified on a PD-10 desalting column, which was equilibratedwith 20% methanol in water. The sample was eluted, frozen andlyophilized to obtain 0.1471 g of crude IMP362 (MH+ 23713).

Synthesis of IMP413 (PEG₃₀-IMP360)

A cartoon diagram of IMP 413 is provided in FIG. 3. For synthesis of IMP413, IMP 360 (0.0103 g) was mixed with 0.1601 g of mPEG-OPTE (30 kDa,Nektar Therapeutics) in 7 mL of 1 M tris buffer at pH 7.81. Acetonitrile(1 mL) was then added to dissolve some suspended material. The reactionwas stirred at room temperature for 4.5 h and then 0.0423 g of TCEP wasadded along with 0.0473 g of cysteine. The reaction mixture was stirredfor 2 h followed by dialysis for two days. The dialyzed material wasfrozen and lyophilized to obtain 0.1552 g of crude IMP413 (MH⁺ 34499).

Example 2 Generation of DDD Module Based on Interferon (IFN)-α2b

Construction of IFN-α2b-DDD2-pdHL2 for Expression in Mammalian Cells

The cDNA sequence for IFN-α2b was amplified by PCR, resulting in asequence comprising the following features, in which XbaI and BamHI arerestriction sites, the signal peptide is native to IFN-α2b, and 6 His isa hexahistidine tag: XbaI---Signal peptide---IFNα2b---6 His---BamHI. Theresulting secreted protein will consist of IFN-α2b fused at itsC-terminus to a polypeptide consisting of SEQ ID NO:2.

(SEQ ID NO: 2) KSHHHHHHGSGGGGSGGGCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

PCR amplification was accomplished using a full length human IFNα2b cDNAclone (Invitrogen Ultimate ORF human clone cat# HORF01Clone ID IOH35221)as a template and the following oligonucleotides as primers:

IFNA2 Xba I Left (SEQ ID NO: 3)5′-TCTAGACACAGGACCTCATCATGGCCTTGACCTTTGCTTTACTGG- 3′ IFNA2 BamHI right(SEQ ID NO: 4) 5′-GGATCCATGATGGTGATGATGGTGTGACTTTTCCTTACTTCTTAAACTTTCTTGC-3′

The PCR amplimer was cloned into the pGemT vector (Promega). ADDD2-pdHL2 mammalian expression vector was prepared for ligation withIFN-α2b by digestion with XbaI and Barn HI restriction endonucleases.The IFN-α2b amplimer was excised from pGemT with XbaI and Barn HI andligated into the DDD2-pdHL2 vector to generate the expression vectorIFN-α2b-DDD2-pdHL2.

Mammalian Expression of IFN-α2b-DDD2

IFN-α2b-DDD2-pdHL2 was linearized by digestion with San enzyme andstably transfected into Sp/EEE myeloma cells by electroporation (see.e.g., U.S. patent application Ser. No. 11/487,215, filed Jul. 14, 2006,incorporated herein by reference). Two clones were found to havedetectable levels of IFN-α2b by ELISA. One of the two clones, designated95, was adapted to growth in serum-free media without substantialdecrease in productivity. The clone was subsequently amplified withincreasing methotrexate (MTX) concentrations from 0.1 to 0.8 μM overfive weeks. At this stage, it was sub-cloned by limiting dilution andthe highest producing sub-clone (95-5) was expanded. The productivity of95-5 grown in shake-flasks was estimated to be 2.5 mg/L using commercialrIFN-α2b (Chemicon IF007, Lot 06008039084) as a standard.

Purification of IFN-α2b-DDD2 from Batch Cultures Grown in Roller Bottles

Clone 95-5 was expanded to 34 roller bottles containing a total of 20 Lof serum-free Hybridoma SFM with 0.8 μM MTX and allowed to reachterminal culture. The supernatant fluid was clarified by centrifugation,filtered (0.2 μM). The filtrate was diafiltered into 1× Binding buffer(10 mM imidazole, 0.5 M NaCl, 50 mM NaH₂PO₄, pH 7.5) and concentrated to310 mL in preparation for purification by immobilized metal affinitychromatography (IMAC). The concentrate was loaded onto a 30-mL Ni-NTAcolumn, which was washed with 500 mL of 0.02% Tween 20 in 1× bindingbuffer and then 290 mL of 30 mM imidazole, 0.02% Tween 20, 0.5 M NaCl,50 mM NaH₂PO₄, pH 7.5. The product was eluted with 110 mL of 250 mMimidazole, 0.02% Tween 20, 150 mM NaCl, 50 mM NaH₂PO₄, pH 7.5.Approximately 6 mg of IFNα2b-DDD2 was purified. FIG. 4 shows the resultsof an anti-IFNα immunoblot and ELISA used to quantify IFNα2b-DDD2.

Characterization of IFN-α2b-DDD2

The purity of IFN-α2b-DDD2 was assessed by SDS-PAGE under reducingconditions (FIG. 5). The Coomassie blue-stained gel shows that the batchproduced from roller bottles (lane 2) was purer than an earlier batch(lane 1). IFN-α2b-DDD2 was the most heavily stained band and accountsfor approximately 50% of the total protein. The product resolves as adoublet with an M_(r) of ˜26 kDa, which is consistent with thecalculated MW of IFN-α2b-DDD2-SP (26 kDa). There is one majorcontaminant with a M_(r) of 34 kDa and many faint contaminating bands.

Example 3 Generation of PEGylated IFN-α2b by DNL

Preparation and Purification of α2b-362 (IFN-α2b-DDD2-IMP362)

A cartoon drawing depicting the structure of α2b-362 having two copiesof IFNα2b coupled to a 20 kDa PEG is provided in FIG. 6. A DNL reactionwas performed by the addition of 11 mg of reduced and lyophilized IMP362in 10-fold molar excess to 2.25 mg (3.5 ml) of IFN-α2b-DDD2 in 250 mMimidazole, 0.02% Tween 20, 150 mM NaCl, 1 mM EDTA, 50 mM NaH₂PO₄, pH7.5. After 6 h at room temperature in the dark, the reaction mixture wasdialyzed against CM Loading Buffer (150 mM NaCl, 20 mM NaAc, pH 4.5) at4° C. in the dark. The solution was loaded onto a 1-mL Hi-Trap CM-FFcolumn (Amersham), which was pre-equilibrated with CM Loading buffer.After sample loading, the column was washed with CM loading buffer tobaseline, followed by washing with 15 mL of 0.25 M NaCl, 20 mM NaAc, pH4.5. The PEGylated product was eluted with 12.5 mL of 0.5 M NaCl, 20 mMNaAc, pH 4.5.

The conjugation process was analyzed by SDS-PAGE with Coomassie bluestaining (FIG. 7A), fluorescence imaging (FIG. 7B) and anti-IFNαimmunoblotting (FIG. 7C). To normalize the samples for direct proteinmass comparison, each fraction eluted from the CM-FF column wasconcentrated to 3.5 mL to match the reaction volume. Under non-reducingconditions, the Coomassie blue-stained gel (FIG. 7A) revealed thepresence of a major band at a M_(r) of 110 kDa (lane 2) in the reactionmixture, which was absent in the unbound (lane 3) or 0.25 M NaCl washfraction (lane 4), but evident in the 0.5 M NaCl fraction (lane 5).Fluorescence imaging (FIG. 7B), which was used to detect the EDANS tagon IMP362, demonstrates that the 110 kDa band contains IMP362 (lanes 2and 5) and the presence of excess IMP362 in the reaction mixture (lane2) and the unbound fraction (lane 3), which does not stain withCoomassie blue. Anti-IFNα immunoblotting (FIG. 7C) confirms theassociation of IFN-α2b with the 110 kDa band (lanes 2 and 5). These datatogether indicate that the DNL reaction results in the site-specific andcovalent conjugation of IMP362 with a dimer of IFN-α2b. Under reducingconditions, which breaks the disulfide linkage, the components of theDNL structures are resolved. The calculated MW of α2b-362 is ˜75 kDa,which matches well the mass of 76,728 Da determined by MALDI TOF. Theobserved discrepancy between the calculated mass and the estimated Mr bySDS-PAGE is due to PEG, which is known to inflate the molecular sizewhen PEGylated products are analyzed by SDS-PAGE or SE-HPLC. Overall,the DNL reaction resulted in a near quantitative yield of a homogeneousproduct that is >90% pure after purification by cation-exchangechromatography.

Preparation and Purification of α2b-413 (IFN-α2b-DDD2-IMP413)

A cartoon drawing depicting the structure of α2b-413 having two copiesof IFNα2b coupled to a 30 kDa PEG is provided in FIG. 8. α2b-413 wasprepared as described immediately above using IMP413 instead of IMP362.

Example 4 Evaluation of the In Vitro Potency of IFN-α2b-DDD2, α2b-362,and α2b-413

In Vitro Anti-Proliferative Assay

IFN-α2b-DDD2 and α2b-362 were assayed for inhibition of growth ofBurkitt's lymphoma (Daudi) cells. Briefly, IFN-α2b standard (ChemiconIF007, Lot 06008039084), IFN-α2b-DDD2 (batch 010207) and α2b-362 (batch010807) were each diluted to 500 pM in RPMI 1640 media supplemented with10% FBS, from which three-fold serial dilutions in triplicate were madein 96-well tissue culture plates (50 μL sample/well). Daudi cells werediluted to 4×10⁵ cells/mL and 50 μL were added to each well (20K/well).The concentration range for each test reagent was 500 pM to 0.008 pM.After 4 days at 37° C., MTS dye was added to the plates (20 μL per well)and after 3 h the plates were read with an Envision plate reader (PerkinElmer, Boston Mass.) at 490 nm. Dose-response curves were generated(FIG. 9) and 50% effective concentration (EC₅₀) values were obtained bysigmoidal fit non-linear regression using Graph Pad Prism software(Advanced Graphics Software, Encinitas, Calif.). The calculated EC₅₀ forIFNα2b-DDD2 and α2b-362 were similar (˜16 μM) and about 5-fold lesspotent than the IFN-α2b standard (EC₅₀-4 pM). In a similar experimentthe α2b-413 had similar potency as α2b-362.

Anti-Viral Assay

Duplicate samples were analyzed in a viral challenge assay usingencephalomyocarditis (EMC) virus on A549 cells by an independentanalytical laboratory (PBL Interferon Source, Piscataway, N.J.). Plateswere stained with crystal violet and the OD was measured byspectrophotometry on a 96-well plate reader following solubilization ofthe dye. The data were analyzed with Graph Pad Prizm software using asigmoidal fit (variable slope) non-linear regression. The anti-viraltiter was determined by comparison of EC₅₀ values with that of an IFNαstandard. The specific anti-viral activities were calculated at 1.2×10⁸U/mg and 8.8×10⁶ U/mg for α2b-362 and α2b-413, respectively.

Example 5 In Vivo Evaluation of α2b-413 and α2b-362

Pharmacokinetics

The study was performed in adult female Swiss-Webster mice (˜35 g).There were 4 different treatment groups of 2 mice each. Each reagent(test and control) was administered at equimolar_protein doses (3 μg ofrhuIFN-α2a, 5 μg of PEGINTRONT™, 11 μg of α2b-362, and 13 μg of α2b-413)as a single bolus i.v. injection. Mice were bled via the retro-orbitalmethod at various time-points (pre-dose, 5-min, 2-, 8-, 24-, 48-, 72-,96-, and 168-h post-injection). The blood was allowed to clot,centrifuged, and the serum was isolated and stored at −70° C. untilassayed for IFN-α concentration and subsequent PK-analysis.

Concentrations of IFN-α in the serum samples were determined using ahuman interferon alpha ELISA kit following the manufacturersinstructions (PBL Interferon Source). Briefly, the serum samples werediluted appropriately according to the human IFN-α standard provided inthe kit. An antibody coupled to the microtiter plate wells capturesinterferon. A second antibody is then used to reveal the boundinterferon, which is quantified by anti-secondary antibody conjugated tohorseradish peroxidase (HRP) following the addition of Tetramethylbenzidine (TMB) substrate. The plates were read at 450 nm, and theresults are shown in FIG. 10.

The PK properties of each agent are summarized in Table 1. As expected,rhIFN-α2a had the most rapid clearance from the blood of injected mice.Its clearance was approximately 3-fold faster than the PEGINTRON™ andmore than 13-fold faster than the DNL-IFN reagents. The PEGINTRON™ wasin turn cleared greater than 4-fold faster than α2b-362 or α2b-413.There was little difference in the elimination rates between α2b-362 andα2b-413.

TABLE 1 Blood Pharmacokinetic Analysis of Interferon-α2b Containing DNLMolecules Administered as Intravenous Injections to Näive Swiss-WebsterMice. IFN Elimination Animal Dose C_(max) T_(1/2α) T_(1/2β) AUC_(0.08→∞)Rate MRT_(0.08→∞) Number (pmol) (pM) (hours) (hours) (h*pM) (l/h) (h)Recombinant Human Interferon-α2a Animal No. 1 160 16,411 0.29 10.537,011 2.34 0.63 Animal No. 2 160 21,835 0.31 7.14 10,147 2.15 0.78 Mean160 19,123 0.30 8.84 8,579 2.25 0.71 PEG-INTRON Animal No. 1 160 87,0900.53 6.29 137,790 0.63 5.42 Animal No. 2 160 105,774 0.43 5.11 150,9050.70 4.79 Mean 160 96,432 0.48 5.70 144,348 0.67 5.11 IFN-α2b-IMP362Animal No. 1 320 60,833 1.72 7.54 379,462 0.16 9.03 Animal No. 2 32097,089 1.43 10.14 570,336 0.17 11.56 Mean 320 78,961 1.58 8.84 474,8990.17 10.30 IFN-α2b-IMP413 Animal No. 1 320 152,923 0.69 12.85 1,012,4700.15 16.75 Animal No. 2 320 100,495 4.03 28.53 1,179,056 0.09 26.56 Mean320 126,709 2.36 20.69 1,095,763 0.12 21.66

In terms of mean residence time (MRT), there is a clear correlation withsize among the various reagents. The 19-kDa rhIFN-α2a had a MRT that was7-fold less than the 31 kDa PEGINTRON™ (0.7 h versus 5.1 h,respectively), which had a 2-fold lower MRT when compared to the 70 kDaα2b-362 (10.3 h). The MRT for the 80 kDa α2b-413 (21.7 h) was 2-foldlonger than α2b-362. Finally, a test for bioequivalence showed that noneof the reagents tested were the same in terms of PK, indicating that thedifferences are genuine (i.e., circulating half-life forα2b-413>α2b-362>PEGINIRON™>rhIFN-α2a).

Anti-Tumor Therapeutic Efficacy

An initial in vivo tumor therapy study demonstrated that theDNL-PEGylated interferons were more potent and longer-lasting comparedto PEGINTRONT™. Eight-week-old female C.B.-17 SCID mice were injectedi.v. with a human Burkitt's lymphoma cell-line (Daudi) at 1.5×10⁷ cellsper animal. There were 10 different treatment groups of 5 mice each.Equivalent units of activity of PEGINTRON™, α2b-362 and α2b-413 wereadministered once every 7 days via s.c. injection in either the left orright flank at three different doses (3500, 7000, and 14000 Units).Therapy commenced 1 day after the Daudi cells were transplanted.

Mice were observed daily for signs of distress and paralysis. They wereweighed weekly. In the event a mouse or mice lost greater than 15% ofits body weight (but <20%) it was weighed every 2 days until it eithergained back its weight to <15% loss or was sacrificed due to >20% loss.Mice were also terminated when hind-limb paralysis developed or if theybecame otherwise moribund.

Survival curves generated from this study are shown in FIG. 11.PEGINTRON™, α2b-362, and α2b-413 all demonstrated significantimprovement in survival when compared to saline control mice (P<0.0016).Except for the 3,500 IU dose of α2b-362, both α2b-413 and α2b-362 weresuperior to PEGINTRON™ when administered at equal activity doses(P≦0.0027). α2b-362 showed more than twice the potency of PEGINTRON™.Doses of 7,000 IU and 3,500 IU of α2b-362 were superior to 14,000 IU(P=0.0016) and 7,000 IU (P=0.0027) doses of PEGINTRON™, respectively.α2b-413 is more than four times as potent as PEGINTRON™ since a 3,500 IUdose of the former was superior to 14,000 IU of the latter (P=0.0027).α2b-413 was significantly better than α2b-362 (P<0.0025) whenadministered at equivalent doses. However, there were no statisticallysignificant differences among the three doses of α2b-413, even thoughthe 14,000 IU dose resulted in a median survival of 60 days incomparison to the 3,500 IU dose and its 46-day median survival(P=0.1255). The in vivo efficacy observed for α2b-362, α2b-413, andPEGINTRON™ thus correlate well with the PK data.

The increased bioavailability of α2b-362 and α2b-413 demonstrated by PKanalysis contributes to the enhanced in vivo anti-tumor potency ofDNL-PEGylated IFNα. In turn, these two factors allow for a less frequentdosing schedule used in tumor therapy. This was demonstrated with asimilar in vivo tumor therapy study as above, in which equal units ofactivity of PEGINTRON™ or α2b-413 were administered with varied dosingschedules. This study was performed in 8-week-old female SCID miceinjected i.v. with Daudi 1.5×10⁷ cells. There were 7 different treatmentgroups of 6-7 mice each. Each reagent (test and control) wasadministered 14,000 IU via a s.c. injection in either the left or rightflank. Therapy was commenced 1 day after the Daudi-cells wereadministered to the mice. One set of mice was dosed once a week for 4weeks (q7dx4), another dosed on a bi-weekly schedule over 8 weeks(q2wkx4), while the third set of mice was dosed once every 3 weeks over12 weeks (q3wkx4). All the mice received a total of 4 injections.

Survival curves generated from this study are shown in FIG. 12. Allanimals that received either form of interferon at any of the variousschedules had significantly improved survival in comparison to salinecontrol mice (P<0.0009). Importantly, all the IFN-IMP413-treated micehad significantly improved survival when compared to those animalstreated at the same schedule with PEGINTRON™ (P<0.0097). Of note, thosemice treated every other week with IFN-IMP413 (q2wkx4) not only hadsignificantly improved survival in comparison to those treated withPEGINTRON™ at the same schedule (MST=>54 days versus 28 days,respectively; P=0.0002), but were also significantly better than thoseanimals treated weekly (q7dx4) with PEGINTRON™ (MST=36.5 days;P=0.0049). Further, survival of mice treated every three weeks withIFN-IMP413 (q3wkx4) was significantly better than those treated withPEGINTRON™ every two weeks (MST=54 days versus 28 days; P=0.002) andapproaches significance when compared to those treated weekly withPEGINTRON™ (P=0.0598).

These studies demonstrate DNL-PEGylation of IFNα2b results in improvedand long-lasting efficacy, allowing for less frequent dosing. Similarenhancements is realized when this technology is applied to othercytokines (such as G-CSF and EPO), growth factors, enzymes, antibodies,immunomodulators, hormones, peptides, drugs, interference RNA,oligonucleotides, vaccines and other biologically active agents.

Example 6 Generation of DDD Module Based on Granulocyte-ColonyStimulating Factor (G-CSF)

Construction of G-CSF-DDD2-pdHL2 for Expression in Mammalian Cells

The cDNA sequence for G-CSF was amplified by PCR resulting in sequencescomprising the following features, in which XbaI and BamHI arerestriction sites, the signal peptide is native to human G-CSF, and 6His is a hexahistidine tag: XbaI---Signal peptide---G-CSF---6 His---BamHI. The resulting secreted protein consisted of G-CSF fused at itsC-terminus to a polypeptide consisting of SEQ ID NO:5.

(SEQ ID NO: 2) KSHHHHHHGSGGGGSGGGCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

PCR amplification was accomplished using a full-length human G-CSF cDNAclone (Invitrogen IMAGE human cat# 97002RG Clone ID 5759022) as atemplate and the following oligonucleotides as primers:

G-CSF XbaI Left (SEQ ID NO: 5)5′-TCTAGACACAGGACCTCATCATGGCTGGACCTGCCACCCAG-3′ G-CSF BamHI-Right (SEQID NO: 6) 5′-GGATCCATGATGGTGATGATGGTGTGACTTGGGCTGGGCAAGGTGGC GTAG-3′

The PCR amplifier was cloned into the pGemT vector. A DDD2-pdHL2mammalian expression vector was prepared for ligation with G-CSF bydigestion with XbaI and Bam HI restriction endonucleases. The G-CSFamplifier was excised from pGemT with XbaI and Bam HI and ligated intothe DDD2-pdHL2 vector to generate the expression vectorG-CSF-DDD2-pdHL2.

Mammalian Expression of G-CSF-DDD2

G-CSF-pdHL2 was linearized by digestion with SalI enzyme and stablytransfected into Sp/EEE myeloma cells by electroporation. Clones wereselected with media containing 0.15 μM MTX. Clone # 4 was shown toproduce 0.15 mg/L of G-CSF-DDD2 by sandwich ELISA.

Purification of G-CSF-DDD2 from Batch Cultures Grown in Roller Bottles

Approximately 3 mg of G-CSF-DDD2 is purified as descried in Example 2.Clone 4 is expanded to 34 roller bottles containing a total of 20 L ofHybridoma SFM with 0.4 μM MTX and allowed to reach terminal culture. Thesupernatant fluid is clarified by centrifugation, filtered (0.2 μM),diafiltered into 1× Binding buffer (10 mM Imidazole, 0.5 M NaCl, 50 mMNaH₂PO₄, pH 7.5 and concentrated. The concentrate is loaded onto aNi-NTA column, which is washed with 0.02% Tween 20 in 1× binding bufferand then 30 mM imidazole, 0.02% Tween 20, 0.5 M NaCl, 50 mM NaH₂PO₄, pH7.5. The product is eluted with 250 mM imidazole, 0.02% Tween 20, 150 mMNaCl, 50 mM NaH₂PO₄, pH 7.5.

Example 7 Generation of PEGylated G-CSF by DNL

A cartoon drawing depicting the structure of G-CSF-413 having two copiesof G-CSF coupled to a 30 kDa PEG is provided in FIG. 13. A DNL reactionis performed by the addition of reduced and lyophilized IMP413 in10-fold molar excess to G-CSF-DDD2 in PBS. After 6 h at room temperaturein the dark, the reaction mixture is purified by immobilized metalaffinity chromatography using Ni-NTA.

Example 8 Generation of DDD Module Based on Erythropoietin (EPO)

Construction of G-CSF-DDD2-pdHL2 for Expression in Mammalian Cells

The cDNA sequence for EPO was amplified by PCR resulting in sequencescomprising the following features, in which XbaI and BamHI arerestriction sites, the signal peptide is native to human EPO, and 6 Hisis a hexahistidine tag: XbaI---Signal peptide---EPO---6 His---BamHI. Theresulting secreted protein consists of EPO fused at its C-terminus to apolypeptide consisting of SEQ ID NO:2.

PCR amplification was accomplished using a full-length human EPO cDNAclone as a template and the following oligonucleotides as primers:

EPO Xba I left (SEQ ID NO: 7)5′-TCTAGACACAGGACCTCATCATGGGGGTGCACGAATGTCC-3′ EPO BamHI Right (SEQ IDNO: 8) 5′-GGATCCATGATGGTGATGATGGTGTGACTTTCTGTCCCCTGTCCTGC AG-3′

The PCR amplifier was cloned into the pGemT vector. A DDD2-pdHL2mammalian expression vector was prepared for ligation with EPO bydigestion with XbaI and Barn HI restriction endonucleases. The EPOamplifier was excised from pGemT with XbaI and Barn HI and ligated intothe DDD2-pdHL2 vector to generate the expression vector EPO-DDD2-pdHL2.

Mammalian Expression of EPO-DDD2

EPO-pdHL2 was linearized by digestion with SalI enzyme and stablytransfected into Sp/EEE myeloma cells by electroporation. Clones wereselected with media containing 0.15 μM MTX. Clones # 41, 49 and 37 eachwere shown to produce ˜0.5 mg/L of EPO by an ELISA using NuncImmobilizer Nickel-Chelate plates to capture the His-tagged fusionprotein and detection with anti-EPO antibody.

Purification of EPO from Batch Cultures Grown in Roller Bottles

Approximately 2.5 mg of EPO-DDD2 is purified by IMAC from 9.6 liters ofserum-free roller bottle culture as described in Example 2. SDS-PAGE andimmunoblot analysis indicate that the purified product constitutesapproximately 10% of the total protein following IMAC (FIG. 14). Underreducing conditions the EPO-DDD2 polypeptide is resolved as a broad bandwith a M_(r) (40-45 kDa) greater than its calculated mass (28 kDa) dueto extensive and heterogeneous glycosylation. Under non-reducingconditions the EPO-DDD2 primarily resolves as a disulfide-linkedcovalent dimer (mediated by DDD2) with a M_(r) of 80-90 kDa.

Example 9 DNL Conjugation of EPO-DDD2 with a Fab-AD2 Module

h679 is a humanized monoclonal antibody that is highly specific for thehapten HSG (histamine-succinyl-glycine). Production of an h679-Fab-AD2module, which is depicted in the cartoon drawing in FIG. 15 (A), hasbeen described previously (Rossi et. al, Proc. Natl. Acad. Sci. USA.2006; 103:6841). A cartoon drawing depicting the dimeric structure ofEPO-DDD2 is provided in FIG. 15 (B). A small-scale preparation ofEPO-679 (EPO-DDD2 x h679-Fab-AD2) was made by DNL. EPO-DDD2 (1 mg) wasreacted overnight with h679-Fab-AD2 (1 mg) in PBS containing 1 mMreduced glutathione and 2 mM oxidized glutathione. The DNL conjugate waspurified by HSG-based affinity chromatography as described previously(Rossi et. al, Proc. Natl. Acad. Sci. USA. 2006; 103:6841). A cartoondrawing depicting the structure of EPO-679 with two EPO moieties andh679-Fab is provided in FIG. 15 (C). Coomassie blue staining of SDS-PAGEgels demonstrated the creation of EPO-679 (FIG. 16). The DNL product,which is resolved as a broad band with a M_(r) of 150-170 kDa undernon-reducing conditions, is highly purified and consists only of thethree constituent polypeptides (EPO, h679-Fd-AD2 and h679 Kappa) asdemonstrated by SDS-PAGE under reducing conditions.

Example 10 Biological Activity of EPO-DDD2 and EPO-679

EPO-DDD2 and EPO-679 were assayed for their ability to stimulate thegrowth of EPO-responsive TF1 cells (ATCC) using recombinant human EPO(Calbiochem) as a positive control. TF1 cells were grown in RPMI 1640media supplemented with 20% FBS without GM-CSF supplementation in96-well plates containing 1×10⁴ cells/well. The concentrations(units/ml) of the EPO constructs were determined using a commercial kit(Human erythropoietin ELISA kit, Stem Cell Research, Cat# 01630). Cellswere cultured in the presence of rhEPO, EPO-DDD2 or EPO-679 atconcentrations ranging from 900 U/ml to 0.001 U/ml for 72 hours. Theviable cell densities were compared by MTS assay using 20 μl of MTSreagent/well incubated for 6 hours before measuring the OD490 in a96-well plate reader. Dose response curves and EC50 values weregenerated using Graph Pad Prism software (FIG. 17). Both EPO-DDD2 andEPO-679 show in vitro biological activity at approximately 10% of thepotency of rhEPO.

Example 11 Generation of PEGylated EPO by DNL

A cartoon drawing depicting the structure of EPO-413 having two copiesof EPO coupled to a 30 kDa PEG is provided in FIG. 18. A DNL reaction isperformed by the addition of reduced and lyophilized IMP413 in 10-foldmolar excess to EPO-DDD2 in PBS. After 6 h at room temperature in thedark, the reaction mixture is purified by immobilized metal affinitychromatography using Ni-NTA.

Example 12 Production of 2-PEG:1-Target Agent Complexes

In alternative embodiments, it is desirable to make PEGylated complexeswith a stoichiometry of 2 PEG moieties to 1 target agent. Such PEGylatedcomplexes are readily made by the methods of Examples 1-3 above, byattaching the PEG moiety to the DDD sequence and the active agent to theAD sequence. A PEGylated complex with a 2:1 stoichiometry of PEG toIFN-α2b is prepared by a modification of the methods of Examples 1-3.The complex exhibits stability in serum and shows interferon activitythat is lower than the PEGylated complex with a 1:2 stoichiometry of PEGto IFN-α2b. However, clearance rate for the bi-PEGylated complex isslower than the clearance rate for the mono-PEGylated complex.

1. A method of PEGylating a therapeutic agent comprising: a) attaching atherapeutic agent to a DDD sequence; b) attaching a PEG moiety to an ADsequence; and c) allowing the DDD sequence to bind to the AD sequence toform a PEGylated entity comprising two therapeutic agent-DDD sequencesand one PEG-AD sequence.
 2. The method of claim 1, wherein the DDD andAD sequences are attached to each other by disulfide bonds.
 3. Themethod of claim 1, wherein the DDD sequence comprises the 44 N-terminalamino acids of PKA RIIα.
 4. The method of claim 1, wherein the DDDsequence comprises SEQ ID NO:2.
 5. The method of claim 1, wherein the ADsequence is from an AKAP protein.
 6. The method of claim 1, wherein theAD sequence comprises SEQ ID NO:1.
 7. The method of claim 1, wherein thePEG moiety is capped at one end with a methoxy group.
 8. The method ofclaim 1, wherein the PEG moiety is linear or branched.
 9. The method ofclaim 1, wherein the therapeutic agent is selected from the groupconsisting of an enzyme, a cytokine, a chemokine, a growth factor, apeptide, an aptamer, hemoglobin, an antibody and an antibody fragment.10. The method of claim 1, wherein the therapeutic agent is a cytokine.11. The method of claim 1, wherein the therapeutic agent is selectedfrom the group consisting of interferon-α, interferon-β, interferon-γ,MIF, HMGB-1 (high mobility group box protein 1), TNF-α, IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-15, IL-16, IL-17, IL-18, IL-19, IL-21, IL-23, IL-24, CCL19, CCL21,MCP-1, RANTES, MIP-1A, MIP-1B, ENA-78, MCP-1, IP-10, Gro-β, Eotaxin,G-CSF, GM-CSF, SCF, PDGF, MSF, Flt-3 ligand, erythropoietin,thrombopoietin, hGH, CNTF, leptin, oncostatin M, VEGF, EGF, FGF, PlGF,insulin, hGH, calcitonin, Factor VIII, IGF, somatostatin, tissueplasminogen activator and LIF.
 12. The method of claim 1, wherein theclearance rate of the PEGylated entity from serum is at least an orderof magnitude slower than the clearance rate of the unPEGylatedtherapeutic agent.
 13. The method of claim 1, wherein the PEG moiety isattached to an AD sequence consisting of a structure selected from thegroup consisting of: (i) IMP350: CGQIEYLAKQIVDNAIQQAGC(SS-tbu)-NH₂ (SEQID NO:1), (ii) IMP360: CGQIEYLAKQIVDNAIQQAGC(SS-tbu)G-EDANS (SEQ IDNO:1), (iii) IMP362: PEG₂₀-CGQIEYLAKQIVDNAIQQAGC(SS-tbu)G-EDANS (SEQ IDNO:1); and (iv) IMP413: PEG₃₀-CGQIEYLAKQIVDNAIQQAGC(SS-tbu)G-EDANS (SEQID NO:1).
 14. The method of claim 1, wherein the therapeutic agent isinterferon (IFN)-α2b, G-CSF or erythropoietin.
 15. The method of claim1, wherein the therapeutic agent attached to the DDD sequence is afusion protein.
 16. A method of PEGylating a therapeutic agentcomprising: a) attaching a therapeutic agent to an AD sequence; b)attaching a PEG moiety to a DDD sequence; and c) allowing the DDDsequence to bind to the AD sequence to form a PEGylated entitycomprising two therapeutic agent-DDD sequences and one PEG-AD sequence.17. The method of claim 16, wherein the DDD and AD sequences areattached to each other by disulfide bonds.
 18. The method of claim 16,wherein the DDD sequence comprises the 44 N-terminal amino acids of PKARIIα.
 19. The method of claim 16, wherein the DDD sequence comprises SEQID NO:2.
 20. The method of claim 16, wherein the AD sequence is from anAKAP protein.
 21. The method of claim 16, wherein the AD sequencecomprises SEQ ID NO:1.
 22. The method of claim 16, wherein the PEGmoiety is capped at one end with a methoxy group.
 23. The method ofclaim 16, wherein the PEG moiety is linear or branched.
 24. The methodof claim 16, wherein the therapeutic agent is selected from the groupconsisting of an enzyme, a cytokine, a chemokine, a growth factor, apeptide, an aptamer, hemoglobin, an antibody and an antibody fragment.25. The method of claim 16, wherein the therapeutic agent is a cytokine.26. The method of claim 16, wherein the therapeutic agent is selectedfrom the group consisting of interferon-α, interferon-β, interferon-γ,MIF, HMGB-1 (high mobility group box protein 1), TNF-α, IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-15, IL-16, IL-17, IL-18, IL-19, IL-21, IL-23, IL-24, CCL19, CCL21,MCP-1, RANTES, MIP-1A, MIP-1B, ENA-78, MCP-1, IP-10, Gro-β, Eotaxin,G-CSF, GM-CSF, SCF, PDGF, MSF, Flt-3 ligand, erythropoietin,thrombopoietin, hGH, CNTF, leptin, oncostatin M, VEGF, EGF, FGF, PlGF,insulin, hGH, calcitonin, Factor VIII, IGF, somatostatin, tissueplasminogen activator and LIF.
 27. The method of claim 16, wherein theclearance rate of the PEGylated entity from serum is at least an orderof magnitude slower than the clearance rate of the unPEGylatedtherapeutic agent.
 28. The method of claim 16, wherein the therapeuticagent is interferon (IFN)-α2b, G-CSF or erythropoietin.
 29. The methodof claim 16, wherein the therapeutic agent attached to the DDD sequenceis a fusion protein.